The long-term objective of this research program is to understand the cell cycle of Escherichia coli. Topics of particular interest are the structure of the bacterial chromosome and how this changes during the replication cycle, the mechanism by which daughter chromosomes are separated and then partitioned to daughter cells, and how completion of the replication cycle is related to subsequent cell division. This information is important for coping with the increasng number of antibiotic resistant bacteria. Dr. Kuempel proposes to study dif-mutants, in which the resolution of chromosome multimers if blocked. This provides a means to study basic properties of the cell cycle. The specific aims are the following: 1) Construct strains with dif inserted at various locations and determine the properties of functional and non-functional sites. This provides a means to study the changes that occur in the terminus region of the chromosome at the end of the replication cycle. 2) Determine where replication forks meet at the end of the replication cycle. This will be done in cells with normal and rearranged chromosomes. The purpose is to determine if functional dif sites must be located where replication forks meet. 3) Develop physical assays for sister chromatid exchange. This will be used to determine the frequency at which daughter chromosomes interact, how this varies at positions around the chromosome, and how this is affected by various recombination mutations. Another purpose of these studies is to test if the primary function of dif is to resolve dimer chromosomes formed by sister chromatid exchange. 4) Characterize the cell cycle of dif mutants. He will determine the basis of the SOS induction that occurs at cell division and characterize the DNA damage that causes this. He will also determine why dif mutants have an elongated interval between termination and cell division (D period), even in cells that are apparently growing normally. This provides a system for studying chromosome partitioning.